Plant Physiol. (1987) 85, 1129-11360032-0889/87/85/1129/08/$0 1.00/0

Calcium Transport in Sealed Vesicles from Red Beet(Beta vulgaris L.) Storage Tissue'I. CHARACTERIZATION OF A Ca2+-PUMPING ATPase ASSOCIATED WITH THE ENDOPLASMICRETICULUM

Received for publication May 5, 1987 and in revised form August 31, 1987

JOHN L. GIANNINI, LYNNE H. GILDENSOPH, INGRID REYNOLDS-NIESMAN, AND DONALD P. BRISKIN*Department ofAgronomy, University ofIllinois, Urbana, Illinois 61801

ABSTRACT

Calcium transport was examined in microsomal membrane vesiclesfrom red beet (Beta vulgaris L.) storage tissue using chlorotetracyclineas a fluorescent probe. This probe demonstrates an increase in fluores-cence corresponding to calcium accumulation within the vesicles whichcan be collapsed by the addition of the calcium ionophore A23187.Calcium uptake in the microsomal vesicles was ATP dependent andcompletely inhibited by orthovanadate. Centrifugation of the microsomalmembrane fraction on a linear 15 to 45% (w/w) sucrose density gradientrevealed the presence of a single peak of calcium uptake which co-migrated with the marker for endoplasmic reticulum. The calcium trans-port system associated with endoplasmic reticulum vesicles was thenfurther characterized in fractions produced by centrifugation on discon-tinous sucrose density gradients. Calcium transport was insensitive tocarbonylcyanide m-chlorophenylhydrazone indicating the presence of aprmary transport system directly linked to ATP utilization. The endo-plasmic reticulum vesicles contained an ATPase activity that was calciumdependent and further stimulated by A23187 (Ca2, A23187 stimulated-ATPase). Both calcium uptake and Ca2+, A23187 stimulated ATPasedemonstrated similar properties with respect to pH optimum, inhibitorsensitivity, substrate specificity, and substrate kinetics. Treatment of thered beet endoplasmic reticulum vesicles with [fr-32P-ATP over short timeintervals revealed the presence of a rapidly turning over 96 kilodaltonradioactive peptide possibly representing a phosphorylated intermediateof this endoplasmic reticulum associated ATPase. It is proposed that thisATPase activity may represent the enzymic machinery responsible formediating primary calcium transport in the endoplasmic reticulum linkedto ATP utilization.

The role of calcium in cell regulation and signal transductionin animal cells has been appreciated for some time (27 andreferences therein). Animal cells maintain low cytoplasmic levelsof calcium by the transport of this ion out of the cell or intovarious intracellular compartments such as the ER and mito-chondria. Upon reception of the proper stimulus (e.g. hormonebinding) the plasma membrane and ER become permeable tocalcium and a transient rise in cytoplasmic calcium level occurs.This rise in cytoplasmic calcium is used in conjunction withother processes (e.g. calmodulin binding, protein kinase activa-tion) as an efficient second messenger system for signal trans-duction (27). An analogous system appears to exist in higher

'Supported by funds from the University of Illinois AgriculturalExperiment Station (Hatch Project No. 15-0327).

plants where calcium is transported across the plasma membrane,into the ER, vacuole, and mitochondria (19 and referencestherein). The induction of calcium release from these structuresby the appropriate stimulus (e.g. hormonal, environmental sig-nals) may cause a transient increase in cytoplasmic calcium andconcomitant signal transduction.With the use of isolated membrane vesicles prepared from

plant cells, mechanisms for calcium transport have been inves-tigated for the tonoplast, ER, and plasma membrane. Calciumuptake at the tonoplast has been recently characterized andshown to be mediated by a Ca2+/H' antiport system indirectlylinked to ATP utilization through the proton electrochemicalgradient established by the tonoplast ATPase (1, 29). Based uponstudies with microsomal membrane vesicles, it has been proposedthat an ATP-dependent calcium pump is associated with theplasma membrane (10, 17), but additional supportive data needsto be provided. Reports on calcium movement into plant ERusing fluorescent (21) and radiotracer (7-9, 29) techniques havesuggested the direct transport of calcium by an ATP-dependentpump, presumably similar to those found for the sarcoplasmicreticulum and ER in animal cells (18). However, the character-ization of a Ca2+-ATPase responsible for this observed calciumtransport at the ER has not been carried out.A recent paper by Lew et al. (21) demonstrated the use of

chlorotetracycline as a fluorescent probe for monitoring themovement of calcium into ER vesicles from zucchini hypocotyl.In the present communication, this probe was used to measurecalcium movement into sealed ER membrane vesicles from redbeet storage tissue and to correlate calcium transport with a Ca",A23187-stimulated ATPase activity. The results of this studyprovide evidence for an ATPase activity responsible for thetransport of calcium into the lumen of the red beet ER.

MATERIALS AND METHODS

Plant Material. Red Beet (Beta vulgaris L., cv Detroit DarkRed) storage roots were purchased commercially. The tops ofthe plants were removed and the storage roots were stored inmoist vermiculite at 2 to 4°C until use. All root tissue used wasstored at least 10 d to ensure uniformity in membrane isolation(24).Membrane Isolation. Sealed membrane vesicles were isolated

according to a modification of the method of Giannini et al.(14). Storage roots were peeled, cut into small cubes and then

2Abbreviations: PMSF, phenylmethylsulfonyl fluoride; BTP, bis-trispropane; DCCD, N'N-dicyclohexylcarbodiimide; DES, diethylstilbestrol;G, gramicidin D; Mes, 4-morpholinoethanesulfonic acid.

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Plant Physiol. Vol. 85, 1987

put into a homogenization medium containing 250 mm sucrose,2 mm EDTA, 2 mM MgSO4, 2 mM Na2ATP, 1% (w/v) BSA(fraction V powder), 0.5% (w/v) polyvinylpyrrolidone (40,000mol wt), 2 mM PMSF,2 15 mM ,B-mercaptoethanol, 4 mM DTE,10% glycerol, and 70 mM Tris/HCl (pH 8.0). DTE, PMSF, and,8-mercaptoethanol were added to the medium just prior to use.The tissue was treated with homogenization medium (1.5:1medium:tissue ratio), by vacuum infiltration, for 5 min at icetemperature before homogenization in a vegetable juice extrac-tor.The homogenate was filtered through four layers ofcheesecloth

and then centrifuged at 13,000g (8500 rpm) for 15 min in aSorvall GSA rotor. The 13,000g pellet was discarded and thesupernatant was centrifuged at 80,000g (32,000 rpm) for 30 minin a Beckman type 35 rotor to obtain a microsomal membranepellet. When used directly in transport assays, the microsomalmembrane pellet was suspended in a suspension buffer contain-ing 250 mm sucrose, 10% (v/v) glycerol, 0.2% (w/v) BSA, 1 mMDTE (added fresh), and 2 mM BTP/Mes (pH 7.0).Sucrose Density Gradient Centrifugation. For centrifugation

on linear sucrose density gradients, the microsomal membranepellet was suspended in 2.0 ml of suspension buffer using adounce-type homogenizer and then layered on a 36 ml linear 15to 45% (w/w) sucrose gradient. The sucrose solutions werebuffered to pH 7.2 with 1 mM Tris/Mes and contained 1 mMDTE. The gradients were centrifuged at 100,000g (25,000 rpm)for 3 h in a Beckman SW 28 rotor and then fractionated into 2ml fractions. The sucrose concentration in the gradient fractionswas determined by refractometry using a Bausche & LombeAbbe refractometer.For the routine preparation of ER membrane vesicles, the

microsomal membrane pellet was suspended in suspension bufferand layered on a discontinuous sucrose density gradient consist-ing of 15 ml of 22% (w/w) sucrose layered over 18 ml of 26%(w/w) sucrose. Both sucrose solutions contained 1 mM Tris/Mes(pH 7.2) and 1 mM DTE. The gradient was centrifuged for 2 hat 100,000g (25,000 rpm) in a Beckman SW 28 rotor and themembranes present at the 22/26% interface were collected. Gra-dient prepared membranes were diluted with an equal volumeof suspension buffer and then centrifuged at 80,000g for 30 min.The ER enriched membrane pellet was suspended in suspensionbuffer at a protein concentration of approximately 2.5 mg/mland either used immediately or frozen under liquid N2 and storedat -80C. Membrane vesicles stored in this manner retainedboth transport and ATPase activity for up to 3 months.Enzyme Assays. Calcium-stimulated phosphohydrolase activ-

ity was determined in a 1 ml reaction volume containing 3.75mM ATP (BTP salt pH 7.25), 3.75 mM MgSO4, 250 mM sorbitol,25 mm BTP/Mes (pH 7.25), 50 AM CaSO4, 0.4 ug/ml A23187,and 40 to 80 ,ug of membrane protein. The release of inorganicphosphate was determined by the method of Ohnishi et al. (23).This assay for the release of inorganic phosphate shows minimalinterference when sorbitol is present. Control assays were run inthe absence of A23 187 and Ca2" and were subtracted to give theCa2+/A23 187 stimulated ATPase activity. Any modifications tothese assay conditions are indicated in "Results and Discussion."Cyt c oxidase and NADH Cyt c reductase assays were per-

formed according to the method of Hodges and Leonard (20).Triton-stimulated UDPase was assayed as described by Naga-hashi and Kane (22).

Optical Measurements of Transport Activity. Proton transportin membrane vesicles was measured by the quenching of quina-crine fluorescence (6, 14). The standard assay for plasma mem-brane vesicles contained 250 mM sorbitol, 25 mM BTP/Mes (pH6.5), 3.75 mm ATP (BTP salt pH 6.5), 3.75 mm MgSO4, 2.5 Mmquinacrine, 100 mMKNO3, and 100,ul aliquots of linear gradientfractions. The assay for tonoplast vesicles contained the samecomponents as for plasma membrane vesicles except KCI re-

placed KNO3 and the assay pH was 7.75. These conditions werefound to be optimal and specific for red beet plasma membrane(13, 14) and tonoplast vesicle (13) proton pumping. Fluorescencemeasurements were carried out at 25°C using a Perkin-Elmermodel 203 Spectrofluorimeter with the excitation monochro-mator set at 430 nm and the emission monochromator set at500 nm.Calcium transport into membrane vesicles was determined

according to the method of Lew et al. (21) with minor modifi-cations. The assay for Ca2+ uptake contained 250 mM sorbitol,25 mm BTP/Mes (pH 7.25), 3.75 mm ATP, 3.75 mM MgSO4, 50mM KCI, 25 Mm CaC12, 7.5 gM (microsomal vesicles), or 20 Mm(ER vesicles) chlorotetracycline (freshly made), and approxi-mately 150 gg of membrane protein. The assay mixture wasallowed to equilibrate for 5 min prior to the addition of ATP toallow stabilization of the fluorescence signal. Release of the Ca2+gradient in the vesicles was accomplished by the addition of 0.3,Mg/ml ofA23 187. Levels of A23 187 greater than 0.5 Mg/ml werefound to interfere with the fluorescence measurement. Fluores-cence measurements were conducted as indicated above with theexcitation monochromator set at 315 nm and the emissionmonochromator set at 555 nm. Fluorescence traces are repre-sentative data from experiments repeated at least three times.Fluorescence data in plots or tables are representative data fromexperiments repeated at least three times. Within each experi-ment, the individual data points represent the mean of twodeterminations. Any variations in these reaction conditions areindicated in 'Results and Discussion."

Phosphorylation and Dodecyl Sulfate Gel Electrophoresis.Phosphorylation reactions were carried out at ice temperature ina 1 ml volume containing 250 mM sorbitol, 50 mM KCI, 2 mg/ml BSA (fraction V powder), 3.75 mM MgSO4, 3.75 mM [Y-32p]ATP (45-70 mCi/mmol), 50 uM CaCl2, 0.4 Mg/ml A23187, 25mM BTP/Mes (pH 7.25) and about 300 Mg ofmembrane protein.The reactions were initiated by the addition of [,y-32P]ATP andquenched by the rapid addition of 25 ml of 10% TCA containing40 mM NaH2PO4, 5 mM Na4P207, and 1 mm Na2ATP. Rapidmixing during the phosphorylation reactions was provided by asmall magnetic stir bar. The phosphorylated protein was collectedby centrifugation at 27,000g (15,000 rpm) for 20 min in a SorvallSS-34 rotor and the resultant pellets were suspended in 8 ml of30 mm HCI. Following centrifugation at 27,000g for 20 min, thephosphorylated protein samples were suspended in 50 M1 of gelelectrophoresis buffer containing 1% (w/w) lithium dodecyl sul-fate, 50 mm Tris-Citrate (pH 2.4), 2% (v/v) ,3-mercaptoethanol,4 M urea, 20% (v/v) glycerol, and 10 Mg/ml pyronin Y (low pHtracking dye) (5). After incubation at room temperature for 10min., aliquots (10 Ml/gel lane) of the phosphorylated sampleswere applied to a 5.6% acrylamide lithium dodecyl sulfate slabgel prepared as previously described (5). Electrophoresis wascarried out at 25 mA per gel for 2.5 h at 20C.

Following electrophoresis, the slab gel was immediately driedonto Whatman No. 1 paper and then subjected to autoradiog-raphy for 36 h against Kodak XAR-5 x-ray film with Cronex'High Plus' intensifying screens at -80°C. Specific phosphoryla-tion conditions are indicated in "Results and Discussion."

Protein Assay. Protein was determined by the method ofBradford (2) using BSA as a standard. The Bradford assay reagentwas filtered just prior to use.

RESULTS AND DISCUSSION

Measurement of Calcium Transport in Microsomal MembraneFractions using Chlorotetracycline. Previous work by Lew et al.(21) demonstrated the feasibility of using chlorotetracycline as a

fluorescent probe in the assay for calcium uptake into ER vesiclesfrom zucchini hypocotyl. This probe forms a complex withdivalent cations such as calcium (1:1 ratio) and exhibits an

1130 GRANNIM ET AL.

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Ca2+-PUMPING ATPasincrease in fluorescence when present in a nonpolar environment(16). Although the measured fluorescence increase for this probereflects the amount of the calcium:probe complex associatedwith membranes, under the appropriate conditions it can serveas a measure of aqueous calcium present in the interior of thevesicle ( 16, 21). Studies were initiated to determine if this probewould also be useful in the assay of calcium uptake in sealedmembrane vesicles isolated from red beet storage tissue. Redbeet microsomal membrane fractions were prepared by a modi-fication of the method of Giannini et al. (14) (omitting theaddition of monovalent salts to the homogenization medium) toyield a fairly even distribution of sealed vesicles (plasma mem-brane, tonoplast, ER) in the final membrane fraction. This wasadvantageous to the present study because it allowed a survey ofthe various sealed vesicle populations that might accumulatecalcium.When red beet microsomal membrane vesicles were assayed

in the presence of chlorotetracycline and 25 Mm CaC12, an ATP-dependent enhancement ofprobe fluorescence was observed (Fig.1). This ATP-dependent increase in fluorescence was collapsedby the addition of 0.3 ug/ml A23187 (calcium ionophore),indicating that the change in chlorotetracycline fluorescencereflects calcium uptake into the vesicles (16, 21). Optimal con-ditions for the measurement of calcium accumulation into mi-crosomal membrane vesicles with this technique occurred when

MICROSOMES ER

+CCCP

z

R23R187

_ RTP RTPV04 V04

TIME

FIG. 1. Measurement of ATP dependent calcium transport in redbeet microsomal membrane and density gradient purified endoplasmicreticulum vesicle fractions using chlorotetracycline fluorescence. Mem-brane vesicles (185 Wg/ml protein) were incubated in the assay media asdescribed in "Materials and Methods." The chlorotetracycline concen-tration in the assay for microsomal membranes and endoplasmic retic-ulum vesicles was 7.5 and 30 uM, respectively. The reaction was initiatedby the addition of 3.75 mM ATP (BTP salt, pH 7.25) and the fluorescenceincrease was collapsed by the addition of 0.3 yg/ml A23187. Whenpresent in the assay, the concentration of sodium vanadate (V04) andcarbonylcyanide m-chlorophenylhydrazone (CCCP) were 100 and 10ums, respectively.

se IN ER OF RED BEET 1131

the concentration of chlorotetracycline and protein werg 7.5 uMand 150 ug/ml, respectively. The ATP-dependent increase inchlorotetracycline fluorescence was completely inhibited by or-thovanadate, suggesting that the observed transport was ener-gized through the action ofATPases which form phosphorylatedintermediates during their reaction cycle (3 and referencestherein). Since the crude microsomal membrane fraction con-tains vesicles derived from several membrane components, thiscould indicate that the calcium transport systems being observedin these assays could reflect either secondary transport systemsassociated with the plasma membrane (driven indirectly by thevanadate inhibited H+-pump) or primary, vanadate sensitivecalcium pumping ATPases associated with the plasma mem-brane (11, 31) or ER (7, 8, 21). The complete inhibition ofcalcium uptake in the presence of orthovanadate; however, wassurprising since our previous studies have indicated that thismembrane preparation technique results in the production ofsealed tonoplast vesicles which have been shown to contain aCa2+/H' antiport driven by the orthovanadate insensitive protonpump (1, 29). In order to address this question and determinethe localization ofthe calcium transport systems being examinedin the fluorescence assay with microsomal vesicles, these mem-branes were further resolved by centrifugation on linear sucrosegradients.

Localization of Calcium Transport Measured by Chlorotetra-cycline Fluorescence. When calcium transport activity was de-termined for the fractions of a linear 15 to 45% (w/w) sucrosegradient, following centrifugation at 100,000g for 3 h, a singlepeak of activity was observed which co-migrated to the samepeak density as NADH Cyt c reductase (KCN insensitive), amarker for the ER (20) (Fig. 2). It should be noted that thisparticular ER marker can also correlate with the presence of theouter envelope of the mitochondria (26), which may explain thesecond peak of activity co-migrating with the mitochondrialmembrane marker at higher densities. Calcium transport activity,as measured with this probe was not associated with the lowdensity peak of nitrate inhibited proton transport characteristicfor tonoplast vesicles (30 and references therein) or the highdensity peak of vanadate inhibited proton transport characteristicfor plasma membrane vesicles (14 and references therein). It alsodid not correspond to the peak densities for Golgi membranesas determined with Triton-stimulated UDPase activity (22) ormitochondrial membranes as determined with Cyt c oxidase(20). From this data, it is apparent that the ATP-dependent,calcium transport observed in the crude microsomal membranefraction reflects the activity of a system associated with the ER.Although sealed plasma membrane and tonoplast vesicles arepresent in this crude membrane fraction ( 14) and these vesicleshave been shown to contain ATP driven calcium transportsystems (1, 1 1, 15, 29), the activity of these transport systemscannot be monitored using chlorotetracycline under our assayconditions. Thus, it would appear that the use of this fluorescentprobe for the assay of calcium transport in isolated membranevesicles may be somewhat selective for the ER. This is especiallyevident with regard to the second paper in this series (15) wherecalcium transport in sealed plasma membrane vesicles can bemeasured using 45Ca2' and cannot be measured in the samesealed vesicles using chlorotetracycline (this paper). While itcould be argued that the levels of Ca2+ used in these assays (25Mm) might inhibit the tonoplast H+-ATPase and hence the drivingforce for indirect Ca2+ transport, previous studies by Schumakerand Sze (29) demonstrated maximal ATP-driven Ca2+ uptake intonoplast vesicles when Ca2+ was present in concentrations ex-ceeding 100 gM. In previous studies by Lew et al. (21), a similarER localization of calcium transport was also observed using thisprobe. However, their membrane preparations isolated fromzucchini hypocotyl did not contain a substantial amount ofsealed plasma membrane vesicles. In order to further characterize

VF - 33%

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1132 GIANNIN

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FIG. 2. Distribution of calcium transport measured by chlorotetra-cycline fluorescence when a red beet microsomal membrane fraction wascentrifuged on a linear sucrose gradient. The sucrose gradient was cen-trifuged at I th,000g for 3 hand then fractionated into 19 equal fractions.Marker enzyme activities were assayed for the fractions as described in"Materials and Methods." Proton transport was assayed as described in"Materials and Methods" and the inhibitor sensitive components wereplotted. These components represent the difference in ionophore revers-ible quench between assays carried out in the presence of KCI and theassay with the inhibitor (13). Calcium transport was determined bymeasuring the initial rate ofchlorotetracycline fluorescence enhancementfor each of the fractions as shown in Figure 1. Sucrose was determinedby refractometry and protein by the Bradford assay (2).

this ER associated calcium transport activity and correspondingCa2"-ATPase activity, a preparative sucrose gradient containinga 22/26% (w/w) intefface was used in the routine preparation ofER enriched membrane fractions.

Characterization of ER Associated Calcium Transport andATPase Activity. Calcium transport, measured by the enhance-ment of chlorotetracycline fluorescence, was substantially en-riched in the vesicles isolated on discontinous sucrose densitygradients (Fig. 1). The optimal measurement of ATP-dependent,calcium transport with the gradient-enriched preparations oc-curred when the chlorotetracycline and protein concentrationswere 20,gm and 150 ~tg/ml, respectively. As with the microsomalmembrane fraction, the fluorescence enhancement was inhibitedby orthovanadate and fully collapsed by A23187. However, theaddition of carbonylcyanide m-chlorophenylhydrazone (CCCP),a proton ionophore, had no effect upon the profile of chloro-tetracycline fluorescence enhancement. This would indicate that

I ET AL. Plant Physiol. Vol. 85, 1987

calcium transport in the red beet ER vesicles was directly linkedto ATP utilization and did not involve proton electrochemicalgradients as an intermediate driving force. A similar conclusionwas reached by Lew et al. (21) for calcium transport in zucchiniER vesicles and Buckhout (7, 8) for ER vesicles from gardencress (Lepidium sativum). Therefore, this calcium transport sys-tem associated with ER vesicles would most likely consist of a

primary calcium transportingATPase ofthe EIE2 class ofenzymewhich characteristically display orthovanadate sensitivity (3 andreferences therein). In order to further explore this possibility,the ATPase activity associated with the ER-enriched, sealedvesicle fraction was characterized and specific properties for bothATPase activity and calcium transport were compared.When assayed in the presence of 3.75 mM Mg:ATP (1:1

concentration ratio), the ATPase activity associated with ERvesicles was stimulated by increasing concentrations of calcium(Fig. 3, upper panel). This stimulation of activity by calciumdiffers from what is observed for the red beet plasma membraneATPase where calcium can act as an inhibitor (4). Calciumstimulation displayed saturation kinetics with a Km of about 7.5AM. If calcium transport was examined under the same condi-tions using chlorotetracycline, a similar kinetic profile was ob-served with a Km of about 5.1 gM. These Km values for calciumstimulation of red beet ER ATPase and calcium dependence ofprobe fluorescence enhancement were fairly close to the Km valuereported for calcium uptake in ER vesicles from garden cress (8).A further increase in ATPase activity in the presence of cal-

cium occurred when A23187 was included in the assays withsealed ER vesicles (Fig. 3, upper panel). This stimulation couldresult from the vesicle ATPase activity being involved in calciumuptake and limited by the extent of the calcium gradient (ther-modynamic feedback). Therefore, a reduction in the gradient by

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20

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Ca 2+ CONCENTRATION (jM)FIG. 3. Effect of calcium concentration on ATPase activity and cal-

cium transport in endoplasmic reticulum vesicles from red beet. Upperpanel: ATPase activity was assayed as described in "Materials andMethods" in the presence of the indicated concentration of Ca2+ (asCaCl2) and the stimulation above the level observed in the basal assaycontaining 3.75 mM ATP, 3.75 mM MgS04, 30 mM BTP/Mes (pH 7.25),50 mm KCI, 250 mM sorbitol, and about 80 4g membrane protein isplotted. The concentration of A23187 (when present) was 0.3 pg/ml.Lower panel: Calcium transport was determined by measuring the initialrate of chlorotetracycline fluorescence increase at the indicated calciumconcentration as described in "Materials and Methods."

- w--

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Qa2+PUMPING ATPase IN ER OF RED BEET 1133

the addition of A23187 would release the ATPase from ther-modynamic limitations and accelerate the rate of ATP hydroly-sis. Although the effects of A23187 could be interpreted in termsof collapsing the calcium gradient, this is complicated by the factthat this carboxylic ionophore mediates calcium movement inexhange for protons (25 and references therein). Since H+-pump-ing ATPase activity could be stimulated by this ionophorethrough collapse of a proton electrochemical gradient, it wouldbecome difficult to correlate A23187 stimulation of ATPaseactivity with calcium uptake if this H+-ATPase activity waspresent as a contaminant. However, the sealed ER vesicles dem-onstrated negligible proton transport activity under the ATPaseassay conditions used for the ER vesicles (A23187 absent) andnegligible stimulation of Ca2+-ATPase activity occurred in thepresence of either CCCP or nigericin when A23187 was absent(data not shown). These observations would suggest that theeffects of A23187 in these assays are related to the collapse ofcalcium gradients in the vesicles so that the A23187 stimulationof ATPase activity would relate to calcium transport. Therefore,this activity was used in subsequent comparisons with calciumtransport measured by chlorotetracycline fluorescence.

0

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FIG. 4. Effect of assay pH on Ca2E, A23187 stimulated-ATPase andcalcium transport in endoplasmic reticulum vesicles from red beet.Standard assays for Ca2", A23187 stimulated ATPase, and calciumtransport were carried out as described in "Materials and Methods" atthe indicated pH values.

The effect of assay pH on calcium transport and Ca2", A23187stimulated-ATPase was determined for the red beet ER vesicles(Fig. 4). Both of these activities displayed a pH optimum at 7.25.In the pH range between 6.75 and 8.0 similar profiles wereobserved for both transport and ATPase, with each showing aslow increase from pH 6.75 to 7.25 followed by a more rapiddecrease to pH 8.0. However, below pH 6.75, calcium transportdecreased to a greater extent than Ca2+, A23187 stimulated-ATPase. This pH optimum for calcium transport is slightly lowerthan the pH optimum of 7.5 observed for calcium transport inER vesicles from zucchini (21) and garden cress (8). It is alsodifferent from the pH optimum observed for the red beet plasmamembrane ATPase (pH 6.5; 4, 14) and tonoplast ATPase (pH7.75; 13, 24).The effect of various inhibitors on calcium transport and Ca2",

A23187 stimulated-ATPase in the sealed red beet ER vesiclesare shown in Table I. Both calcium transport and Ca2+, A23187stimulated-ATPase were inhibited by orthovanadate, and DESbut insensitive to sodium azide (N3). In general, calcium trans-port was inhibited to a greater degree than the ATPase activityin the presence of these compounds. When calcium transport

z

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0

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100

20

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INa3VO41 (lln)FIG. 5. Calcium transport and Ca2l, A23 187 stimulated-ATPase ac-

tivity in the presence of increasing concentrations of sodium orthovan-adate. Assays for Ca2+, A23 187 stimulated-ATPase and calcium transportwere carried out as described in "Materials and Methods" with theindicated concentration of sodium orthovanadate.

Table I. Effect of Various Inhibitors on Calcium Transport and Ca2", A23187-StimulatedATPase Activity inSealed ER Vesiclesfrom Red Beet Storage Tissue

Treatment Fluoresence Increasea Ca2+, A23187-stimulated ATPasea% min Amol Pi/h-mg protein

Controlb 7.46 (I0 )C 2.23 (10)C+ 50 AM DCCD 2.78 (37.3) 1.46 (65.0)+ 30 jiM DES 1.89 (25.0) 1.05 (47.0)+ 1 mm NaN3 7.72 (103) 2.47 (110)+ 100 AM Na3VO4 0.0 (0) 0.0 (0)+ 50 mM KNO3 (-50 mM 1.90 (25.5) 1.69 (76.0)KCI)

+ 50 mM KNO3 6.80 (91.0)+ 50 mM KNO3, 50 mM 2.00 (26.8)K/Mes (-50 mM KCI)a Data for the rate of fluorescence increase are a representative data set for experiments repeated at least

three times with similar results. Data for ATPase activity represent the mean of three separate experi-ments. b The control assay, for the measurement of calcium transport by chlorotetracycline fluorescence,contained 250 mM sorbitol, 25 mM BTP/Mes (pH 7.25), 3.75 mm ATP (BTP salt, pH 7.25), 3.75 MgSO4, 50mM KCI, 25 AM CaCl2, 25 AM chlorotetracycline and about 300 lAg of membrane protein. The control assayfor Ca2+, A23187 stimulated-ATPase only differed from the assay for calcium transport by the absence ofchlorotetracycline and a reduction in the amount of membrane protein present (80 Mg/assay). c Values inparenthesis indicate the percentage of the control assay value.

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Plant Physiol. Vol. 85, 1987

and Ca2+, A23187 stimulated-ATPase were examined in thepresence of increasing concentrations of orthovanadate, full in-hibition of both activities was achieved at 100 uM (Fig. 5). Thispattern of inhibition is different from that displayed by the redbeet plasma membrane ATPase ( 14, 24) and its associated protontransport activity (14) which are less sensitive to this inhibitor.These activities associated with plasma membrane vesicles arenot completely inhibited at vanadate concentrations as high as200 uM.With the exception of the effect of DCCD, the response of the

calcium transport system in red beet ER vesicles to inhibitorswas similar to that observed by Lew et al. (21) for calciumtransport in zucchini vesicles. In contrast to our observed inhi-bition of calcium transport by DCCD, these workers found thatthis compound stimulated calcium transport. An additional dif-ference in the ER calcium transport systems for these two prep-arations occurred in their response to nitrate. In the zucchinivesicle system, calcium transport was stimulated by 50 mMnitrate when assayed in the presence of 50 mM KCI and it wasconcluded that nitrate represented an effective counterion forcharge compensation during calcium transport (21). In contrast,nitrate was found to inhibit calcium transport by 75% in the redbeet ER vesicles when measured in the absence of KCI (Table I).This inhibition could be substantially reduced in the presence of50 mM KCI and a comparison to an assay where KCI was replacedby K/Mes indicated that the chloride anion was responsible forthis restoration of activity in the presence of nitrate. These resultscould imply a more effective role for chloride than nitrate incharge compensation during calcium transport in the vesicles.However, an alternative explanation could be that these anionsexert differential effects upon probe binding to the membrane.Calcium transport and Ca2+, A23 187 stimulated-ATPase were

examined in the presence of various phosphate compounds todetermine their effectiveness as substrates for these activities(Table II). Both calcium transport and ATPase in the red beetER vesicles showed a strong preference for ATP as the substratefor transport and hydrolysis. In contrast, calcium transport inER vesicles from garden cress occurred in the presence of otherphosphate substrates (ADP, GTP, ITP) but at levels not exceed-ing 40% of the activity observed with ATP (7).When calcium transport and Ca2", A23 187 stimulated ATPase

were measured in the presence of various concentrations ofMg:ATP (1:1 concentration ratio), simple Michaelis-Menten ki-netic profiles were observed for both of these activities (Fig. 6).When the data were transformed in terms of the Hanes-Woolflinear arrangement of the Michaelis-Menten equation, the re-spective Km values for calcium transport and Ca2", A23187stimulated-ATPase were found to be 0.4 and 0.38 mM. Thesevalues are in good agreement, but higher than those reported for

Table II. Specificity ofCalcium Transport and Ca2, A23187Stimulated-A TPasefor Phosphorylated Compounds in Sealed ER

Vesiclesfrom Red Beet Storage Tissue

Substrate' Fluoresence Ca24, A23187Substratea Increase Stimulated-ATPase

%1min g~mol Pi/h.- mg pro-%/min tein

ATP 4.27b 3.02bGTP 0.0 0.0UTP 0.0 0.0AMP 0.0 0.01CDP 0.0CMP 0.0

a Phosphate substrates were present in the standard assays at a concen-tration of 3.75 mM. b Data for fluorescence increase are representativedata set for experiments repeated at least three times with similar results.Data for ATPase activity represent the mean oftwo separate experiments.

0.

4cI,,.

.4C.

S1

cm

z-

Ea

enw

4

wzU,W

0

-j

-

E

be

Mg:ATP CONCENTRATION (mM)FIG. 6. Calcium transport and Ca2+, A23187 stimulated-ATPase ac-

tivity as a function of Mg:ATP concentration for endoplasmic reticulumvesicles from red beet. When plotted according to the Woolf-Hanestransformation of the Michaelis-Menten equation: S/V = K.in V.DB + 1/V,.[S] the Km for Ca2+, A23187 stimulated ATPase was 0.38 mM whilethe Km for calcium tanport was 0.4 mM.

calcium transport in ER vesicles from zucchini (0.1 mM) (21)and garden cress (0.073 mM) (8).

Phosphorylation of the ER Vesicle Fraction. The observationthat both calcium transport and Ca2+, A23 187-stimulatedATPase were sensitive to low concentrations of orthovanadatewould imply that this calcium transporting ATPase forms aphosphorylated intermediate during its reaction cycle (3 andreferences therein). In order to examine this possibility, ERvesicles were phosphorylated at ice temperature using Ly32p]ATP under the same conditions as the assay for Ca2+, A23 187-stimulated ATPase and the phosphorylated proteins were ana-lyzed by lithium dodecyl sulfate gel electrophoresis (Fig. 7). Thisgel system was used since it provides optimal conditions (lowtemperature, low pH) for maintaining the labile phosphorylatedintermediates of transport ATPases during electrophoresis (5 andreferences therein). Phosphorylation of the ER membranes for20 s revealed the presence of two major radioactive bands withmol wt of 96 and 35 kD (lanes 3 and 4). The phosphorylation ofboth of these bands was enzymic in nature since the labeling wasnot present when the ER membrane sample was heat denatured(boiled for 5 min) prior to phosphorylation with [y-'2P]ATP(lanes 1 and 2). The radioactivity present at the bottom of thegel most likely represents residual ['y-'2P]ATP or [32P] inorganicphosphate not removed by the washing steps during samplepreparation since it remained associated with the lanes repre-senting the heat denatured ER membrane sample.

Phosphorylation of 96 kD band demonstrated rapid turnoversince the labeling of this polypeptide was reduced by a 40 s chasewith a 100-fold excess of unlabeled ATP following phosphoryl-ation for 20 s at ice temperature (lanes 5 and 6). The labelingassociated with this band was also decreased when the phospho-rylated sample was incubated with 0.25 M hydroxylamine (lanes7 and 8) suggesting that the protein phosphate bond is an acylphosphate linkage (5 and references therein). These propertiesand molecular size (i.e. 90-115 kD) of the 96 kD polypeptideare typical for phosphorylated intermediates associated with theE,E2 type of transport ATPase (3, 5 and references therein) andwould suggest that this polypeptide might represent the catalyticsubunit of the ER Ca2+-ATPase.

In contrast, the phosphorylation associated with the 35 kDpolypeptide was unaffected by both the chase with a 100-foldexcess of unlabeled ATP and the treatment with 0.25 M hydrox-ylamine. This would indicate that the labeling associated withthis band did not show the rapid turnover characteristic ofenzyme reaction intermediates and did not involve the formationof an acyl-phosphate type bond (3, 5). Therefore, the phospho-rylation of this band may be the result of protein kinase activityalso associated with the ER vesicle fraction (5).

1134 GIANNINI ET AL.

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La2PUMPING ATPase IN ER OF RED BEET 1135

1 2 3 4 5 6 7 8

FIG. 7. Gel autoradiograph of the phos-phorylated protein associated with a redbeet endoplasmic reticulum vesicle frac-tion. Phosphorylation and electrophoresiswere carried out as described in "Materialsand Methods." The dried gel was placed

K against x-ray film for 36 h at -80°C with aCronex 'high plus' intensifying screen. Ap-proximately 60 jug of membrane proteinwas applied per slab gel lane. Lanes I and2, membrane sample boiled 5 min prior tophosphorylation; lanes 3 and 4, 20-s phos-phorylation; lanes 5 and 6, 20-s phospho-rylation followed by a 40-s chase with a100-fold excess of unlabeled ATP; lanes 7and 8, 20-s phosphorylation with the

e quenched proteins treated with 0.25 M hy-droxylamine (pH 5.2) for 30 min on iceprior to the 30 mm HCI wash.

GENERAL DISCUSSION AND CONCLUSION

The results of this study demonstrate the presence of an ATP-dependent, primary calcium transport system associated with theER of red beet storage tissue, when chlorotetracycline was usedas a fluorescent probe for calcium accumulation in sealed vesicles(16, 21). Since it is reasonable to assume that the active site forATP is located on the outside surface of the ER (facing thecytoplasm), this system would be involved in the transport ofcalcium from the cytoplasm to the lumen of the ER. Our resultssuggest that this direct coupling of ATP utilization to calciumtransport at the ER of red beet involves the action of a calciumpumping ATPase associated with this membrane. This activityin sealed ER vesicles is calcium dependent and further stimulatedby the calcium ionophore, A23187. This stimulation by A23 187would presumably reflect the removal of any thermodynamicinhibition on the ATPase by calcium gradients produced as aresult of transport in the sealed vesicles. The direct involvementof this ER associated ATPase activity in driving calcium trans-port is also supported by the observation that both calciumtransport and ATPase activity (Ca2", A23187) demonstrate anumber of similar properties with respect to inhibitor sensitivity,substrate specificity, pH optimum, and substrate kinetics.The properties of this calcium pumping ATPase associated

with the ER of red beet suggest that it may have a role inmaintaining low cytoplasmic levels of calcium important to thefunction of this cation as a second messenger (19 and referencestherein). The Km values for Ca2", A23187-stimulated ATPaseactivity and Ca2" transport (measured by chlorotetracycline flu-orescence) are well within the range of those reported for Ca2+transport at the ER (2.5 FM; Ref. 8), tonoplast (15 jLAm; Ref. 29;41.7 ,uM; Ref. 1), and a putative plasma membrane preparation(15 AM; Ref. 10). This would be consistent with the proposedrole of this ER calcium pump in participating with these othersystems in reducing cytoplasmic calcium levels. It is believedthat upon induction by the proper stimulus, the stored Ca2"

within the ER and other storage compartments can be releasedcausing a transient rise in cytoplasmic Ca2l levels with a concom-itant response due in part to the action ofcalmodulin and proteinkinases ( 19 and references therein). Recent evidence suggests thatinositol- 1,4,5-triphosphate may have an important role in stim-ulating this release of calcium from internal storage sites such asthe ER (12, 28).The sensitivity of calcium transport and Ca2", A23 1 87-stimu-

lated ATPase in red beet ER vesicles to low concentrations oforthovanadate strongly suggested that this calcium pumpingATPase forms a covalent phosphorylated intermediate duringATP hydrolysis. Treatment of the ER membrane fraction with[y-32P]ATP revealed the presence of two phosphorylated pep-tides. One peptide with a mol wt of about 35 kD demonstratedproperties (i.e. lack of turnover) consistent with a protein kinaseorigin. However, a labeled polypeptide with a mol wt of about96 kD demonstrated the rapid turnover consistent with it repre-senting the reaction intermediate of an enzyme such as the ERATPase. The protein-phosphate linkage for this peptide wasshown to be an acyl-phosphate bond, typical of the 'high energy'reaction intermediates of E,E2 type transport ATPases, by itssensitivity to hydroxylamine (3, 5, 18). Furthermore, the molec-ular size of this phosphorylated polypeptide is similar to thephosphorylated intermediates associated with transport enzymessuch as the animal cell Na+,K+-ATPase, sarcoplasmic reticulumCa2+-ATPase, gastric mucosal H+,K+-ATPase and the plantplasma membrane ATPase (3, 5, 18 and references therein).Work currently in progress also indicates that the phosphoryla-tion ofthis 96 kD peptide is calcium dependent and demonstratesa sensitivity to inhibitors similar to the Ca2+, A23 187-stimulatedATPase and calcium transport (I Niesman, DP Briskin, unpub-lished results). The association of a 96 kD phosphorylated inter-mediate with the ER Ca2+-ATPase of plant cells would indicatethat this enzyme may be similar in structure and properties tothe Ca2+-ATPase associated with the sarcoplasmic reticulum inanimal muscle cells (18 and references therein). Our future www.plantphysiol.orgon July 5, 2020 - Published by Downloaded from

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1136 GIANNIN ET AL.

studies will focus on confirming the association of this phospho-rylated intermediate with the ER Ca2+-ATPase from red beetstorage tissue and the potential use of this catalytic phosphoryl-ation as a means to understand the reaction mechanism of thisenzyme.

LITERATURE CITED

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2. BRADFORD MM 1979 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye bind-ing. Anal Biochem 72: 248-254

3. BRISKIN DP 1986 Plasma membrane H+-transporting ATPase: role in potas-sium ion transport? Physiol Plant 68: 159-163

4. BRISKIN DP, RJ POOLE 1983 Characterization of a K+-stimulated adenosinetriphosphatase associated with the plasma membrane of red beet. PlantPhysiol 71: 350-355

5. BRISKIN DP, RJ POOLE 1983 Plasma membrane ATPase of red beet forms aphosphorylated intermediate. Plant Physiol 71: 507-512

6. BRISKIN DP, WR THORNLEY, RE WYSE 1985 Membrane transport in isolatedvesicles from sugarbeet taproot. I. Isolation and characterization of energy-dependent, H+-transporting vesicles. Plant Physiol 78: 865-870

7. BUCKHOUT TJ 1983 ATP-dependent Ca2+ transport in endoplasmic reticulumisolated from roots of Lepidium sativum L. Planta 159: 84-90

8. BUCKOUT TJ 1984 Characterization of Ca2 transport in purified endoplasmicreticulum membranes from Lepidium sativum L. roots. Plant Physiol 76:962-967

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10. DIETER P, D MARMt 1980 Calmodulin activation of plant microsomal Ca2+-uptake. Proc NatI Acad Sci USA 77: 7311-7314

1 1. DIETER P, D MARMt 1981 A calmodulin-dependent, microsomal ATPase fromcorn (Zea mays L.). FEBS Lett 125: 245-248

12. DROBAK BK, IB FERGUSON 1985 Release of Ca24 from plant hypocotylmicrosomes by inositol-1,4,5-triphosphate. Biochem Biophys Res Commun130: 1241-1246

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Plant Physiol. Vol. 85, 1987

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19. HEPLER PK, RO WAYNE 1985 Calcium and plant development. Annu RevPlant Physiol 36: 397-439

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22. NAGAHASHI G, AP KANE 1982 Triton stimulated nucleoside diphosphataseactivity: subcellular localization in corn root homogenates. Protoplasma 112:167-173

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24. POOLE RJ, DP BRISKIN, Z KRATKY, RM JOHNSTONE 1984 Density gradientlocalization of plasma membrane and tonoplast from storage tissue ofgrowing and dormant red beet. Characterization of proton-transport andATPase in tonoplast vesicles. Plant Physiol 74: 549-556

25. PRESSMAN BC 1976 Biological applications ofionophores. Annu Rev Biochem45: 501-550

26. QUAIL PH 1979 Plant cell fractionation. Annu Rev Plant Physiol 30: 425-48427. RASMUSSEN H 1981 Calcium and cAMP as synarchic messengers. Wiley Press,

New York28. RINCON M, WF Boss 1987 myo-Inositol triphosphate mobilizes calcium from

fusogenic carrot (Daucus carota L.) protoplasts. Plant Physiol 83: 395-39829. SCHMAKER KS, H SZE 1985 A Ca24/H+ antiport system driven by the proton

electrochemical gradient of a tonoplast H+-ATPase from oat roots. PlantPhysiol 79: 1111-1117

30. SZE H 1985 H+-translocating ATPases: advances using membrane vesicles.Annu Rev Plant Physiol 36: 175-208

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